The main focus of the laboratory in 2005-6 was in elucidating structural mechanisms of lipid and phophoinositide signaling and protein trafficking, with an emphasis on mechanisms relevant to AIDS and type II diabetes and on fundamental mechanisms important to eukaryotic cell biology.[unreadable] [unreadable] Protein trafficking:[unreadable] [unreadable] 1. Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination, and contains an intrinsic ubiquitin ligase (E3) activity, all of which require an N-terminal A20 zinc finger and an immediately C-terminal helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5 ? resolution shows that Rabex-5:ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin binding domain, an inverted ubiquitin interaction motif (IUIM), that binds with ~29 micromolar affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ~22 micromolar affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc finger diaromatic patch mediates ubiquitin ligase activity by directly recruiting a ubiquitin-loaded ubiquitin conjugating enzyme.[unreadable] [unreadable] 2. The Endosomal Sorting Complex Required for Transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 ? resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEVdomain, the Vps28 C-domain, and other domains project to bind their partners.[unreadable] [unreadable] 3. The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1? resolution reveals two curved_?-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.[unreadable] [unreadable] Lipid signaling and trafficking:[unreadable] [unreadable] 1. Although receptor-mediated regulation of small G-proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. As part of a collaboration with Marcelo Kazanietz, Univ. of Pennsylvania, we have identified the Rac-GAP beta2-chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase C-gamma and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes beta2-chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the beta2-chimaerin C1 domain as well as by PLCgamma1 RNAi, thus defining beta2-chimaerin as a novel DAG effector. On the other hand, GAP-deficient beta2-chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of beta2-chimaerin significantly extended the duration of Rac activation by EGF, suggesting that beta2-chimaerin serves as a mechanism that self-limits Rac activity in response to EGFR activation. These results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine-kinase receptor via a PKC-independent mechanism.[unreadable] [unreadable] 2. Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). In collaboration with Will Prinz and Sumana Raychaudhuri, NIDDK, we showed that this transport requires oxysterol-binding protein (OSBP)-related proteins (ORPs), which are a large family of conserved lipid-binding proteins. A representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. The findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.